Reflective microspheres for ultrasonic imaging

ABSTRACT

The present invention provides a method of contrast enhanced ultrasonic diagnostic imaging comprising administering to a subject a contrast enhancing amount of spheres or particles comprising a matrix enclosing a contrast agent which reflects sound waves, and generating an ultrasonic image of said subject wherein the matrix comprises a polymer held together by chemical bonds and wherein the polymer is selected from the group consisting of acrylates, polystyrene and derivatives thereof.

This application is a Continuation of application Ser. No. 08/445,945, filed May 22, 1995, U.S. Pat. No. 5,670,135, which is a Continuation of application Ser. No. 08/361,466, filed Dec. 22, 1994, now U.S. Pat. No. 5,618,514, which is a Continuation of application Ser. No. 08/186,096, filed Jan. 25, 1994, now abandoned, which is a Continuation of application Ser. No. 08/043,982, filed Apr. 7, 1993, now abandoned, which is a Continuation of application Ser. No. 07/888,305, filed May 27, 1992, now abandoned, which is a Continuation of application Ser. No. 07/693,031, filed Apr. 30, 1991, now abandoned, which is a Continuation of application Ser. No. 07/278,326, filed Nov. 30, 1988, now abandoned, which is a Continuation of application Ser. No. 06/775,047, filed Aug. 15, 1985, now abandoned, which is a 371 of application serial No. PCT/SE84/00437, filed Dec. 20, 1984.

BACKGROUND

The invention relates to response particles, preferably spheres, and their use as a diagnostic and contrast agent.

In diagnostic medicine, contrast agents are today being used primarily in X-ray diagnostics where an increased contrast effect is obtained during examination of, for example, internal organs, such as the kidneys, the urinary tract, the digestive tract, the vascular system of the heart (angiography), etc. This contrast effect is based upon the fact that the contrast agent itself is less permeable to X-rays than the surrounding tissue, as a result of which a different blackening of the X-ray plate is obtained.

X-raying implies certain radiation hazards, but during angiography the complication risk is associated in particular with the use of contrast agents.

In recent years, a number of new methods have been introduced in diagnostic radiology. One such method goes by the name NMR (Nuclear Magnetic Resonance) which provides information not only about the distribution of the water content in a specific tissue (in contradistinction to radiology which merely gives a measure of the transmissibility of the X-rays in a specific tissue), but also about the chemical tissue structure which is different in normal and pathological tissue.

In the NMR method, a strong and homogeneous magnetic field is applied across the tissue to be examined. By studying the so-called relaxation times of the protons of the molecules present, especially the protons of the water, it is possible to produce, via comprehensive and complex computer calculations, a visual image of the structure of the tissue concerned.

There is, however, an interest in being able to make a differential diagnosis between pieces of tissue having a high density of blood vessels and, alternatively, tissue having a low density of vessels. Such a situation which has considerable clinical interest, comprises the localisation of tumours which, in their periphery, have a higher density of vessels as compared with normal tissue.

One useful method in the context is to inject into the vascular system some type of particles responsive to a magnetic field and showing changes in the above-mentioned relaxation times.

These magnetically responsive particles interfere with the above-mentioned homogeneous magnetic field in that there is formed, around each individual particle, a field gradient which in its turn changes the relaxation times.

Put more simply, this means that "black holes" are formed around each particle which may be visualised and thus give an impression of the vessel density in the tissue in question.

In another diagnostic method, use may be made of the movability of magnetically responsive particles in a tissue. The basic principle of this method may be studied according to the following: If magnetically responsive particle are introduced into a magnetic field, the particles will align themselves in the direction of the field lines. If the field is removed or shut down, the magnetically responsive particles will change their position in response to processes in the tissue. The duration of this change may, however, vary between different tissues and also in dependence upon the localisation of the particles within the tissue in question. This variability of the response of the magnetic material may be utilised diagnostically. If magnetically responsive particles are administered to a patient, the distribution of the particles in different organs can be determined by means of a sensitive magnetometer capable of detecting the above-mentioned changes (Nature (1983) 302, 336).

Ultrasonics is another visualisation technique in which sound-waves are reflected differently against different types of tissue, depending upon the acoustic impedance of these tissues. Also in this respect, there is an interest in being able to use some type of contrast agent in order to obtain an amplification of specific organs. Particles of different types have here been shown to provide changed echo effects and a changed resolution associated therewith (J. Acoust.Soc.Am. (1983) 74, 1006).

It is also possible to use magnetically responsive particles having a Curie point of about 42° C. for use at hyperthermia. In this instance, the magnetically responsive particles are retained during the treatment of the hyperthermia by a magnetic field, but the moment the tissue temperature exceeds the Curie point, the particles disappear from the tissue because the magnetic responsiveness disappears at this temperature.

By labelling the particles with some gamma-radiating nuclide (for example technetium-99m) it is possible to localise the particles by means of a gamma camera and thereby also to combine the examination with some of the other techniques referred to above.

When using particles within any of the above-mentioned ranges, it is desired, in order to achieve optimal conditions, to be able to vary the amount of magnetically or otherwise responsive material, without affecting on that account the pharmacodynamic and circulatory characteristics of the particles. To be able to do this, one must use a technique which implies enclosing the responsive material in a matrix, preferably a matrix of spherical shape, and the matrix should per se satisfy the following criteria:

biocompatible

biologically degradable

nonimmunogenic.

A matrix of this type normally is built up of some type of polymers which are held together by chemical bonds. Different types of polymers are available for making such matrices. However, the selection of polymers will be very limited if the above-mentioned criteria of the matrix are to be fulfilled.

One type of polymers that has proved useful in these contexts are the carbohydrates, especially those who are included in the body as a natural constituent.

Endogenous carbohydrates are represented by starch and glycogen, but also dextran may be included in this group because of its prolonged use as a blood substituent in medical service.

The production of a carbohydrate matrix satisfying these criteria is described in PCT/SE82/00381, PCT/SE83/00106 and PCT/SE83/00268.

Another type of polymers that have proved to satisfy the said criteria are polyamino acids, such as proteins of the type albumin. The production of polyamino acid matrices is disclosed in U.S. Pat. No. 4,247,406.

Further types of polymers are represented by synthetic polymers, such as acrylates, polystyrene etc. The production of matrices from synthetic polymers is well documented in literature.

It is in this connection extremely advantageous if covalent cross-linking of the polymers can be avoided in the production of a useful matrix. For example, covalently cross-linked carbohydrate matrices have been found to produce transformed cells, in the form of granuloma, when used on humans (Am.Surg. (1955) 142, 1045).

When using covalently cross-linked proteins, there is a risk of immunological reactions because the resulting derivatised protein is not recognised by the body's immunity system as a protein belonging to the body. There are, however, for specific systems no alternatives to the covalent cross-linking, especially when using synthetic polymers or combinations of different polymers and cross-linking agents in order to obtain a useful system. As an example, it is possible to cross-link acrylic polymers with starch and, alternatively, to cross-link starch with acrylates.

Another useful possibility which is described in literature is the production of larger particles from smaller particles. For example, it is possible to produce, from 0.5 μm particles, conglomerates of larger particles, for example in the range of about 10 μm.

Another factor of importance to the injection of spheres into the vascular system is that the spheres have a size that prevents them from getting stuck in the capillary system of different tissues during the first passage. To prevent this, the particle must have a diameter below 1 μm (Chem.Pharm.Bull. (1975) 23, 1452) and a surface structure of hydrophilic character.

When particles are injected into the vascular system, all particles will have collected after a given period of time in the liver or spleen (the RES system) because the normal function of these organs is to purify the blood of foreign particles. At present, there is only one method described which is capable of collecting particles to organs other than those mentioned above, and this is by utilising magnetically responsive particles or spheres.

This is of particular interest in the context of this invention because spheres containing magnetically responsive substances can be used and be made to stop in different tissues by means of the outer magnetic field. When the magnetically responsive particles then are stuck in the desired tissue, the tissue in question can simply be visualised by means of the NMR method or some of the other techniques referred to above.

DESCRIPTION OF THE INVENTION

The invention relates to responsive particles, preferably spheres, and their use as a diagnostic and contrast agent. The invention shows that it is possible to utilise spheres as contrast agents, the responsive material being enclosed within a matrix. The responsive material may consist of, for example, magnetite particles enclosed in the form of discrete particles of varying size, or in the form of complexed ions.

One conceivable matrix for use in the context of this invention consists of carbohydrates that have been stabilised by crystallization, which means that the type of chemical bonds holding the polymeric network together is not covalent in character, mainly hydrogen bonds, van der Waals forces or, in some cases, ion bonds.

As carbohydrate, use may be made of all conceivable variants, including carbohydrates of varying molecular weight and/or substituted or otherwise derivatised carbohydrates. For example, it may be mentioned that it is possible to produce and use magnetically responsive carbohydrate particles in which the carbohydrate is of starch origin and low-molecular of the type glucose, maltose, dextrins etc., and successively increasing molecular weight up to native potato starch having a molecular weight of several millions. The same molecular weight range is applicable to other carbohydrate groups, such as dextran or glycogen.

Another matrix for use in the complex of this invention may consist of polyamino acids, such as the protein albumin in which the matrix is stabilised by heating and the cohering forces are not covalent in character, of the type hydrophobic interactions, hydrogen bonds, van der Waals forces or ion bonds. In a manner similar to what has been stated above, it is also possible to use synthetic polymers or combinations as matrix.

The following Example should not be regarded as restrictive and merely serves to illustrate the main features of the invention.

EXAMPLE

Dextran spheres having a size of about 1 μm with enclosed magnetite particles (size 10-20 nm) were suspended in human serum. The relaxation times of the solution were measured with an NMR apparatus (Praxis II, Alnor Instrument AB, Nykoping) and compared with the relaxation times for the same serum without magnetically responsive dextran spheres. The following values of T1 and T2, respectively, were obtained.

    ______________________________________                                                           T1 (ms)                                                                              T2 (ms)                                                ______________________________________                                         Serum without particles:                                                                           1660    400                                                Serum with particles: conc.:                                                   0.05 mg/ml          1342    109                                                 0.1 mg/ml          1306    82.2                                                0.2 mg/ml          1147    52.6                                                0.5 mg/ml          968     30.7                                                1.0 mg/ml          813     24.0                                                2.0 mg/ml          688     19.9                                                4.0 mg/ml          691     22.9                                               ______________________________________                                     

What is claimed is:
 1. A method of contrast enhanced ultrasonic diagnostic imaging comprising administering to a subject a contrast enhancing amount of spheres or particles comprising a matrix enclosing a contrast agent which reflects sound waves, and generating an ultrasonic image of said subject wherein the matrix comprises a polymer held together by chemical bonds and wherein the polymer is selected from the group consisting of acrylates, polystyrene and derivatives thereof.
 2. The method of claim 1 wherein the polymer is covalently crosslinked.
 3. The method of claim 1 wherein the polymer comprises an acrylic polymer crosslinked with starch.
 4. The method of claim 1 wherein the polymer comprises starch crosslinked with acrylates.
 5. The method of claim 1 wherein the spheres or particles have a surface structure of hydrophilic character.
 6. The method of claim 1 wherein the spheres or particles have a diameter in the range of 0.01-1000 μm.
 7. The method of claim 1 wherein the spheres or particles have a diameter less than 1 μm.
 8. The method of claim 1 wherein the particles are built up of conglomerates of smaller particles or spheres.
 9. The method of claim 8 wherein the size of the conglomerates is about 10 μm.
 10. The method of claim 8 wherein the size of the smaller particles or spheres is about 0.5 μm.
 11. The method of claim 1 wherein an ultrasonic image is generated of the reticuloendothelial system, including the liver and spleen, of said subject.
 12. The method of claim 1 comprising the further step of collecting particles to organs by additionally utilizing magnetically responsive particles or spheres and an outer magnetic field.
 13. A method of contrast enhanced ultrasonic diagnostic imaging which comprises administering to a subject a contrast enhancing amount of spheres or particles comprising non-immunogenic matrix material enclosing solid particles of a diagnostic agent, and generating an enhanced ultrasound image.
 14. A method as claimed in claim 13, wherein said particles are magnetically responsive particles.
 15. A method as claimed in claim 14, wherein said magnetically responsive particles are magnetite particles.
 16. A method as claimed in claim 13, wherein the matrix has been stabilized by means of non-covalent bonds.
 17. A method as claimed in claim 13, wherein the matrix material comprises carbohydrates and their derivatives, polyamino acids or synthetic polymers.
 18. A method as claimed in claim 17, wherein the matrix, when it is a synthetic polymer, has been stabilized by means of covalent bonds.
 19. A method as claimed in claim 13, wherein the spheres or particles have diameters of 0.01-1000 μm.
 20. A method as claimed in claim 19, wherein the diameters are less than 1 μm.
 21. A method as claimed in claim 13, wherein the spheres or particles per se have been built up of conglomerates of smaller particles or spheres. 